Staphylococcal aureus (S. aureus) fibronectin-binding protein (Fnb) is a surface-associated multifunctional receptor responsible for specific reversible binding to human proteins such as fibronectin, fibrin and fibrinogen. Such binding allows the microorganism to effectively attach and subsequently invade and colonize the human host during surgeries, vascular injuries, etc. Fnb has been evaluated as a potential candidate for inclusion in immunogenic compositions to prevent S. aureus infections. Immunization with recombinant Fnb and the generation of functionally active antibodies against this protein potentially can prevent the initial attachment of S. aureus to human tissues, and therefore, prevent infection. Recent studies, however, indicate that antibodies generated in mice, rabbits and humans do not inhibit binding of wild-type Fnb to human fibronectin and fibrinogen. Rather, they induce binding of Fnb to these human proteins, and thus, enhance bacterial adherence to the host tissue. This indicates that reversible binding to human proteins serves only as an initial phase in the process of staphylococcal adhesion to the host tissue.
In a recent study, it was demonstrated that staphylococcal fibronectin-binding protein A (FnbA) serves as a substrate for the human enzyme called plasma transglutaminase (Matsuka et al. 2003). This is a novel function, previously unknown for FnbA. Plasma transglutaminase (also known as Factor XIIIa) is an enzyme that catalyzes covalent (irreversible) cross-linking of a very limited number of human proteins (Table 1) resulting in the formation of high molecular mass homo- and heteropolymers. Factor XIII is a member of the transglutaminase family of enzymes that catalyze the formation of isopeptide bond(s) either within or between polypeptide chains. Factor XIII circulates in the blood and is therefore considered to be an extracellular enzyme, whereas tissue transglutaminases (TG) (e.g., liver TG, keratinocyte TG, epidermal TG, prostate TG and erythrocyte TG) are located inside the cells, and therefore, act as intracellular enzymes (Aeschlimann et al. 1994). Distinct transglutaminases recognize the same protein as substrate, but often with a different affinity and/or specificity. Overall, the substrate specificity for Factor XIIIa is more stringent than for tissue transglutaminases (Gorman et al. 1981; Gorman et al. 1984; Fesus et al. 1986).
Cross-linking reactions catalyzed by Factor XIIIa are important steps in various normal physiological reactions including blood coagulation, wound healing, and fibrinolysis. Factor XIIIa-catalyzed protein cross-linking takes place via formation of covalent bonds between specific glutamine (Gln) and lysine (Lys) amino acid residues. It has been demonstrated that FnbA can be readily cross-linked to human fibronectin and fibrin by Factor XIIIa (Matsuka et al. 2003). Thus, upon immunization with FnbA, FnbA undergoes immediate covalent (irreversible) cross-linking to fibronectin and fibrin. This formation of an irreversible complex of an antigen with human proteins very likely compromises the immune response and leads to the production of antibodies that lack inhibitory/neutralizing activity.
Thus, there is a need to identify the specific reactive amino acid residues (Gln and Lys) within wild-type staphylococcal fibronectin-binding protein that are directly involved in Factor XIIIa-catalyzed covalent cross-linking with human proteins, and substitute those residues to produce an altered form of Fnb that has reduced reactivity to coagulation Factor XIIIa and will effectively inhibit cross-linking and irreversible binding to fibronectin and fibrin.